Abstract:

The present invention is based on the discovery that the cytoxicity of
anti-desmocollin 2 (DSC2) antibodies can be used for treating various
cancers including lung, colon, pancreatic, prostate, breast, gastric or
liver cancers. Specifically, the present invention provides antibodies
against DSC2 that have effector function. Furthermore, the present
invention provides methods and pharmaceutical compositions that comprise
anti-DSC2 antibody as an active ingredient for damaging DSC2-expressing
cells via the effector function of the antibody.

wherein the CDR1, CDR2, and CDR3 are separated by framework amino acid
sequences.

12. The pharmaceutical composition of claim 6, wherein the polynucleotide
is contained in a vector.

13. A method for damaging DSC2-expressing cells, comprising the steps
of:a) contacting DSC2-expressing cells with the antibody of claim 1;
andb) damaging the DSC2-expressing cells through the effector function of
the antibody that bound to the cell.

14. An immunogenic composition for inducing antibody with effector
function against DSC2-expressing cells in a subject, wherein the
composition comprises, as an active ingredient, DSC2, an immunologically
active fragment thereof, or a polynucleotide that expresses DSC2 or the
immunologically active fragment.

15. A method for inducing the production of antibody that has effector
function against DSC2-expressing cells in a subject, wherein the method
comprises administering to the subject DSC2, an immunologically active
fragment thereof, or a cell or a polynucleotide that expresses DSC2 or
the immunologically active fragment.

21. The polypeptide of claim 17, wherein the antibody further comprises
the Fc region of human IgG1.

22. A polynucleotide encoding the polypeptide of claim 16.

23. A vector comprising the polynucleotide of claim 22.

24. A host cell comprising the polynucleotide of claim 22 or a vector
containing the polynucleotide.

25. A method for producing the polypeptide of claim 16, comprising
culturing a cell that expresses the polypeptide, and recovering the
polypeptide from the cell culture.

Description:

[0001]This application claims the benefit of U.S. Provisional Application
Ser. No. 60/680,609 filed May 12, 2005, the contents of which are hereby
incorporated by reference in their entirety.

TECHNICAL FIELD

[0002]DSC2 has been found to be specifically expressed in various cancer
cells including lung, colon, pancreatic, prostate, breast, gastric, and
liver cancer cells. The present invention relates to antibodies against
desmocollin 2 (DSC2) having effector function, and the use of the
antibodies in methods and compositions for damaging DSC2-expressing cells
via the effector function of the anti-DSC2 antibodies.

BACKGROUND ART

[0003]Lung cancer is one of the most common lethal human tumors.
Non-small-cell lung cancer (NSCLC) is the most common form, accounting
for nearly 80% of lung tumors (American Cancer Society, Cancer Facts and
Figures 2001, Am. Chem. Soc. Atlanta, 2001). The majority of NSCLCs are
not diagnosed before advanced stage, and thus the overall 10-year
survival rate has stayed as low as 10%, despite recent advances in
multimodality therapies (Fry et al, Cancer 86: 1867-76, 1999). Currently,
chemotherapy using platinum is considered to be a fundamental therapy for
NSCLCs. However, the therapeutic action of pharmaceutical agents has not
progressed beyond the point of being able to prolong the survival of
advanced NSCLC patients to a certain extent (Non-small Cell Lung Cancer
Collaborative Group, Bmj 311: 899-909, 1995). A number of targeting
therapies are being investigated, including those that use tyrosine
kinase inhibitors. However, to date, promising results have been achieved
only in a limited number of patients, and in some patients, therapeutic
effects have accompanied severe side effects (Kris et al., Proc Am Soc
Clin Oncol 21: 292a (A1166), 2002).

[0004]Colorectal carcinoma is a leading cause of cancer deaths in
developed countries. Specifically, more than 130,000 new cases of
colorectal cancer in USA are reported each year. Colorectal cancer
represents about 15% of all cancers. Of these, approximately 5% are
directly related to inherited genetic defects. In spite of recent
progress in therapeutic strategies, prognosis of patients with advanced
cancers remains very poor. Although molecular studies have revealed the
involvement of alterations in tumor suppressor genes and/or oncogenes in
carcinogenesis, the precise mechanisms still remain to be elucidated.

[0005]Pancreatic cancer has one of the highest mortality rates of any
malignancy, and the 5-year-survival rate of patients is 4%. 28,000 people
are diagnosed as having pancreatic cancer each year, and nearly all of
these patients die of their disease (Greenlee R T et al., Cancer
statistics, 2001. CA Cancer J Clin 51: 15-36, 2001). The poor prognosis
of this malignancy is a result of the difficulty of early diagnosis and
poor response to current therapeutic methods (Greenlee R T et al., Cancer
statistics, 2001. CA Cancer J Clin 51: 15-36, 2001; Klinkenbijl J H et
al., Ann Surg 230: 776-82, and discussion 782-4, 1999). In particular,
currently no tumor marker is identified that allows reliable screening at
an early, potentially curative stage of the disease.

[0006]Prostate cancer (PRC) is one of the most common malignancies in men
and represents a significant worldwide health problem. It is the second
most frequent cause of cancer death in USA (Greenlee R T et al., Cancer
statistics, 2001 CA Cancer J Clin 51: 15-36, 2001). Incidence of PRC is
steadily increasing in developed countries according to the prevalence of
Western-style diet and increasing number of senior population. Increasing
number of patients also die from this disease in Japan due to adoption of
a Western life style (Kuroishi T, Epidemiology of prostate cancer.
Klinika 25: 43-8, 1995). Currently, the diagnosis of PRC is based on an
increased level of the serum prostate specific antigen (PSA). Early
diagnosis provides an opportunity for curative surgery. Patients with
organ confined PRC are usually treated and approximately 70% of them are
curable with radical prostatectomy (Roberts W W et al., Urology 57:
1033-7, 2001; Roberts S G et al., Mayo Clin Proc 76: 576-81, 2001). Most
of patients with advanced or relapsed disease are treated with androgen
ablation therapy due to the androgen-dependent initial growth of PRC.
Although most of these patients initially respond to androgen ablation
therapy, the disease eventually progresses to androgen-independent PRC,
at which point the tumor is no longer responsive to androgen ablation
therapy.

[0007]One of the most serious clinical problems in the treatment for PRC
is that this androgen-independent PRC is unresponsive to any other known
therapies. Thus, clarifying the mechanism of androgen-independent growth
and establishing new therapies other than androgen ablation therapy
against PRC are urgent issues for the management of PRC.

[0008]Breast cancer, a genetically heterogeneous disease, is the most
common malignancy in women. An estimation of approximately 800,000 new
cases is reported each year worldwide (Parkin D M, et al., CA Cancer J
Clin 49: 33-64, 1999). Mastectomy is the first concurrent option for the
treatment of this disease. Despite surgical removal of the primary
tumors, relapse at local or distant sites may occur due to
micrometastasis that is undetectable at the time of diagnosis (Saphner T,
et al., J Clin Oncol 14: 2738-46, 1996). Cytotoxic agents are usually
administered as adjuvant therapy after surgery aiming to kill those
residual or premalignant cells.

[0009]Treatment with conventional chemotherapeutic agents is often
empirical and is mostly based on histological tumor parameters, and in
the absence of specific mechanistic understanding. Target-directed drugs
are therefore becoming the bedrock treatment for breast cancer. Tamoxifen
and aromatase inhibitors, two representatives of its kind, have been
proved to achieve great responses when used as adjuvant or
chemoprevention in patients with metastasized breast cancer (Fisher B et
al. J Natl Cancer Inst 90: 1371-88, 1998; Cuzick J, Lancet 360: 817-24,
2002). However, the drawback is that only patients who express estrogen
receptors are sensitive to these drugs. Further, regarding their side
effects, long term tamoxifen treatment may cause endometrial cancer as
well as deleterious effect of bone fracture in the postmenopausal women
in aromatase prescribed patients (Coleman R E Oncology 18(5 Suppl 3):
16-20, 2004). Owing to the emergence of side effects and drug resistance,
it is obviously necessarily to search novel molecular targets for
selective smart drugs on the basis of characterized mechanisms of action.

[0010]Gastric cancer is a leading cause of cancer death in the world,
particularly in the Far East, with approximately 700,000 new cases
diagnosed worldwide annually. Surgery is the mainstay in terms of
treatment, because chemotherapy remains unsatisfactory. Gastric cancers
at an early stage can be cured by surgical resection, but prognosis of
advanced gastric cancers remains very poor.

[0011]Hepatocellular carcinoma (HCC) is one of the most common cancers
worldwide and its incidence is gradually increasing in Japan as well as
USA (Akriviadis E A et al., Br J Surg 85(10): 1319-31, 1998.). Although
recent medical advances have made great progress in diagnosis, a large
number of patients with HCCs are still diagnosed at advanced stages and
their complete cures from the disease remain difficult. In addition,
since patients with hepatic cirrhosis or chronic hepatitis have a high
risk to HCCs, they may develop multiple liver tumors, or new tumors even
after complete removal of initial tumors. Therefore, development of
highly effective chemotherapeutic drugs and preventive strategies are
matters of pressing concern.

[0012]Research aiming at the elucidation of carcinogenic mechanisms has
revealed a number of candidate target molecules for anti-tumor agents.
For example, the farnesyltransferase inhibitor (FTI) is effective in the
therapy of Ras-dependent tumors in animal models (Sun J. et al.,
Oncogene.; 16:1467-73, 1998.). This pharmaceutical agent was developed to
inhibit growth signal pathways related to Ras, which is dependant on
post-transcriptional farnesylation. Human clinical trials where
anti-tumor agents were applied in combination with the anti-HER2
monoclonal antibody trastuzumab with the aim of antagonizing the
proto-oncogene HER2/neu have succeeded in improving clinical response,
and improved the overall survival rate of breast cancer patients.

[0013]Tyrosine kinase inhibitor STI-571 is an inhibitor which selectively
deactivates bcr-abl fusion protein. This pharmaceutical agent was
developed for the therapy of chronic myeloid leukemia, where the constant
activation of bcr-abl tyrosine kinase has a significant role in the
transformation of white blood cells. Such pharmaceutical agents are
designed to inhibit the carcinogenic activity of specific gene products
(O'Dwyer M E & Druker B J. Curr Opin Oncol.; 12:594-7, 2000.). Today,
gene products with promoted expression in cancer cells are usually
potential targets for the development of novel anti-tumor agents.

[0014]Another strategy for cancer therapy is the use of antibodies which
bind to cancer cells. The following are representative mechanisms of
antibody-mediated cancer therapy:

[0015](I) Missile therapy: in this approach, a pharmaceutical agent is
bound to an antibody that specifically binds to cancer cells, and the
agent then specifically acts on the cancer cells. Through this method the
pharmaceutical agent intensively acts on the cancer cells, therefore,
even agents with strong side effects can be used with less side effects.
In addition to pharmaceutical agents, there are also reports of
approaches where precursors of pharmaceutical agents, enzymes which
metabolize the precursors to an active form, and so on are bound to the
antibodies;

[0016](II) The use of antibodies which target functional molecules: this
approach inhibits the binding between growth factors and cancer cells
using, for example, antibodies that bind to growth factor receptors or
growth factors. Some cancer cells proliferate depending on the activity
of growth factors. For example, cancers dependent on epithelial growth
factor (EGF) or vascular endothelial growth factor (VEGF) are known. For
such cancers, inhibiting the binding between a growth factor and the
cancer cells can be expected to have a therapeutic effect; and

[0017](III) Antibody cytotoxicity: antibodies that bind to some kinds of
antigens on cancer cells can exert cytotoxicity to the cancer cells.
These types of antibodies have itself a direct anti-tumor effect.
Antibodies that display cytotoxicity to cancer cells are gaining
attention as antibody agents expected to be highly effective against
tumors.

[0018]The object and features of the present invention will become more
fully apparent when the following disclosure of the invention is read in
conjunction with the accompanying figures and examples. However, it is to
be understood that the following disclosure is of preferred embodiments,
and not restrictive of the invention or other alternate embodiments of
the invention.

[0020]The terms "isolated" and "purified" used in relation with a
substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates
that the substance is substantially free from at least one substance that
may else be included in the natural source. Thus, an isolated or purified
antibody refers to antibodies that is substantially free of cellular
material such as carbohydrate, lipid, or other contaminating proteins
from the cell or tissue source from which the protein (antibody) is
derived, or substantially free of chemical precursors or other chemicals
when chemically synthesized. The term "substantially free of cellular
material" includes preparations of a polypeptide in which the polypeptide
is separated from cellular components of the cells from which it is
isolated or recombinantly produced. Thus, a polypeptide that is
substantially free of cellular material includes preparations of
polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight)
of heterologous protein (also referred to herein as a "contaminating
protein"). When the polypeptide is recombinantly produced, it is also
preferably substantially free of culture medium, which includes
preparations of polypeptide with culture medium less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the polypeptide is
produced by chemical synthesis, it is preferably substantially free of
chemical precursors or other chemicals, which includes preparations of
polypeptide with chemical precursors or other chemicals involved in the
synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry
weight) of the volume of the protein preparation. That a particular
protein preparation contains an isolated or purified polypeptide can be
shown, for example, by the appearance of a single band following sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein
preparation and Coomassie Brilliant Blue staining or the like of the gel.
In a preferred embodiment, antibodies of the present invention are
isolated or purified.

[0021]An "isolated" or "purified" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or substantially
free of chemical precursors or other chemicals when chemically
synthesized. In a preferred embodiment, nucleic acid molecules encoding
antibodies of the present invention are isolated or purified.

[0022]The terms "polypeptide", "peptide", and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
including antibodies. The terms apply to amino acid polymers in which one
or more amino acid residue is a modified residue, or a non-naturally
occurring residue, such as an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to naturally
occurring amino acid polymers.

[0023]The term "amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics that
similarly functions to the naturally occurring amino acids. Naturally
occurring amino acids are those encoded by the genetic code, as well as
those modified after translation in cells (e.g., hydroxyproline,
γ-carboxyglutamate, and O-phosphoserine). The phrase "amino acid
analog" refers to compounds that have the same basic chemical structure
(an a carbon bound to a hydrogen, a carboxy group, an amino group, and an
R group) as a naturally occurring amino acid but have a modified R group
or modified backbones (e.g., homoserine, norleucine, methionine,
sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic"
refers to chemical compounds that have different structures but similar
functions to general amino acids.

[0024]Amino acids may be referred to herein by their commonly known three
letter symbols or the one-letter symbols recommended by the IUPAC-IUB
Biochemical Nomenclature Commission.

[0025]The terms "gene", "polynucleotides", "nucleotides", "nucleic acids",
and "nucleic acid molecules" are used interchangeably unless otherwise
specifically indicated and are similarly to the amino acids referred to
by their commonly accepted single-letter codes.

[0026]The term "antigen" refers to proteins that have the binding ability
to a corresponding antibody and induce the antigen-antibody reaction in
vivo. On the other hand, the term "immunogen" refers to the group of
proteins among the antigens that further have the ability to induce the
production of antibody in vivo.

[0027]Antibodies" and "immunoglobulins" are glycoproteins having the same
structural characteristics. While antibodies exhibit binding specificity
to a specific antigen, immunoglobulins include both antibodies and other
antibody-like molecules, for which antigen specificity has not been
defined. Polypeptides of the latter kind are, for example, produced at
low levels by the lymph system and at increased levels by myelomas.

[0028]Herein, the term "antibody" refers to molecules belonging to any
class or subclass of immunoglobulins. Depending on the amino acid
sequence of the constant domain of their heavy chains, immunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be
further divided into subclasses (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins are
called α, δ, ε, γ, and μ, respectively. The
subunit structures and three-dimensional configurations of different
classes of immunoglobulins are well known.

[0029]The term "antibody" herein includes both monoclonal and polyclonal
antibodies. The term also includes modified antibodies that retain the
specific antigen-binding ability of the original antibody, for example,
antibodies bound to other molecules, chimeric antibodies (humanized
antibodies etc.), antibodies wherein one or more amino acids therein are
substituted, deleted, added, or inserted, and the like. Furthermore, the
term is intended to encompass fragments of antibodies so long as they
retain their specific binding ability to its antigen. Such fragments
include, for example, Fv, Fab, F(ab')2, scFv, etc., however, the
present invention is not restricted thereto and includes much smaller
portions of the antibody that still possess the specific binding ability
of the original antibody.

[0030]The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies, i.e.,
the individual antibodies comprising the population are identical except
for possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations, which typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. In addition to their specificity, the monoclonal antibodies are
advantageous in that they can be synthesized by hybridoma culture,
uncontaminated by other immunoglobulins. Thus, the modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to be
construed as requiring production of the antibody by any particular
method. For example, the monoclonal antibodies to be used in accordance
with the present invention can be made by the hybridoma method first
described by Kohler and Milstein, (Nature 256:495-7, 1975), or can be
made by recombinant DNA methods (Cabilly et al., Proc Natl Acad Sci USA
81:3273-7, 1984).

[0031]The monoclonal antibodies herein specifically include "chimeric"
antibodies or immunoglobulins, in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a particular
antibody class or subclass, while the remainder of the chain(s) is
identical with or homologous to corresponding sequences in antibodies
derived from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies, so long as they
exhibit the desired biological activity (Cabilly et al., Proc Natl Acad
Sci USA 81:3273-7, 1984; Morrison et al., Proc Natl Acad Sci USA
81:6851-5, 1984). Most typically, chimeric antibodies or immunoglobulins
comprise human and murine antibody fragments, generally human constant
and mouse variable regions.

[0032]The "effector function" refers to the cytotoxicity of an antibody.
The cytotoxicity is usually involved with the Fc region of the antibody,
however, the present invention is not restricted thereto and "antibodies
with effector function" encompasses all kinds of antibodies that cause
damage to cells on which the antigen of the antibodies are expressed.

[0033]Specifically, antibody-dependent cell-mediated cytotoxicity (ADCC;
also referred to as antibody-dependent cellular cytotoxicity),
complement-dependent cytotoxicity (CDC), and neutralizing activity are
known as antibody effector functions, and are detailed below. These
biological activities of the antibody can independently damage cells,
however, in practice, they function in composite in living cells. Thus,
an antibody of the invention may have one, two or all of these effector
functions. Preferable effector functions herein are either, ADCC, CDC, or
both.

(1) Antibody Dependent Cell-mediated Cytotoxicity (ADCC)

[0034]Antibody dependent cell-mediated cytotoxicity (ADCC) refers to a
cell damaging reaction which is caused on a target cell via the action of
effector cell and antibody, in particular IgG class antibodies. Hence the
amount of antibody required for causing this effect is quite small, this
cytotoxic function is considered to be important where only weak antibody
production reaction is caused, like in tumors, autoimmune diseases, etc.
It is known that ADCC is an important mechanism in cancer therapies using
antibodies (Clynes R A, et al., Nature Med 6: 443-6, 2000). For example,
ADCC is reported to be an important effector mechanism for the treatment
of cancer using anti-CD20 chimeric antibody (Cartron G, et al., Blood 99:
754-8, 2002). Thus, when applying the present invention for cancer
therapies, the effector function of ADCC becomes particularly important.

[0035]Cells involved in this reaction are called effector cells and
acquire cytotoxicity by binding to the antigen bound antibodies. Example
of such cells includes lymphocytes (T cells, NK cells, etc.),
macrophages, polymorphonuclear leukocyte (neutophils), K cell, and the
like. These cells carry receptors, called Fc receptors that bind to the
Fc region of antibodies bound on the cell surface through an antigen. It
is known that each of the Fc receptors specifically recognize and bind to
the Fc region of a specific class and/or subclass of the immunoglobulins.
For example, cells comprising Fc receptors specific to the Fc region of
the immunoglobulin class IgG include T cells, NK cells, neutrophils, and
macrophages, and are activated by the Fc region of IgG class antibodies
to exert cytotoxicity against cells to which these antibodies have bound.

[0036]ADCC can be classified based on the involved effector cell to
IgG-dependent macrophage-mediated cytotoxicity (ADMC) and IgG-dependent
NK-cell-mediated cytotoxicity (narrow sense ADCC). Herein, the term ADCC
is used in the broad sense and encompasses ADMC, where macrophages
function as the effector cell.

[0037]Antibody ADCC is known to be an important mechanism of anti-tumor
effects caused in a living body, particularly important in cancer
therapies that use antibodies (Clynes R A, et al., Nature Med 6: 443-6,
2000). For example, a close relationship between the therapeutic effect
of anti-CD20 antibody chimeric antibodies and ADCC has been reported
(Cartron G, et al., Blood 99: 754-8, 2002). Thus, ADCC is particularly
important among the antibody effector functions in the present invention.

[0038]At present, the mechanism of ADCC is roughly explained as follows:
first, an antibody binds to the target cell, then an effector cell
recognizing the Fc region of the antibody, binds to the antibody. The
effector cell, which is bridged to the target cell via the antibody bound
to the cell surface, is thought to induce target cell apoptosis by
transmitting some sort of lethal signal to the target cell.

(2) Complement-Dependent Cytotoxicity (CDC)

[0039]The Fc region of antibodies of an antibody-antigen complex is known
to activate the complement system. The complements involved in this
system are sequentially activated through enzymatic reaction or binding
with other activated complements and form molecules that show biological
activities, such as induction of histamine release, acting as chemotactic
factors for neutrophils and macrophages, opsonin activity, etc. Among
these activated molecules, C5b-9 membrane attack complex (MAC) damage
viral particles and cell membranes independent of effector cells. MAC
exerts a strong binding affinity for cell membranes, and the molecule
bound on a cell membrane opens a hole, making it easy for water to flow
in and out of the cell. As a result, the cell membrane gets destabilized,
or the cell is destroyed through the change in osmotic pressure. The
biological activity caused by an activated complement or complex of the
complements only extends to a region close to the antigen-antibody
complex which activated the complement system. In particular, the
function of lysing cells to which the antibody variable region has been
bound is defined as CDC.

[0040]Further, the pathway to activate the complement system has been
revealed to differ depending on the immunoglobulin class of the antibody
inducing the pathway. For example, among the human antibodies, IgM and
IgG activate the classical pathway. On the other hand, IgA, IgD, and IgE
do not activate this pathway.

(3) Neutralizing Activity

[0041]Some antibodies are known to have the function of depriving
infectivity of pathogens and/or activity of toxins. Such neutralization
of pathogens and toxins can be achieved through the binding of the
antigenic variable region of an antibody to an antigen included in the
pathogens or toxin. Sometimes, the neutralization is known to require not
only the antibody but complement mediation to deprive a virus of its
infectivity. Thus, in case of using an antibody with neutralizing
activity which requires complement mediation in therapy and such, the Fc
region, essential for activating the complement system, is necessary in
addition to the antigenic variable region.

[0042]Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. In case of conflict,
the present specification, including definitions, will control.

DETAILED DESCRIPTION OF THE INVENTION

[0043]Conditions required for destroying cancer cells using effector
function of an antibody are, for example, as follows: [0044](a)
expression of large numbers of antigenic molecules on the membrane
surface of target cancer cells; [0045](b) uniform distribution of
antigens within target cancerous tissues; and [0046](c) lingering of
antigens bound to antibodies on the cell surface for a long time.

[0047]More specifically, for example, antigens recognized by antibodies
are required to be expressed on the surface of the cell membrane of
target cancer cell. In addition, it is preferable that the ratio of
antigen-positive cells is as high as possible in cells forming cancerous
tissues. For example, an ideal situation may be where all target cancer
cells in the objective tissue are positive to that antigen. When an
antigen existing only in a portion of a cancer cell population of the
objective tissue is used as the target, no clinical therapeutic effect
may be expected for antibodies against that antigen. Usually, the higher
the expression of the antigen used as the target on the cell surface, the
stronger the effector functions to be expected.

[0048]Furthermore, it is also important that antibodies bound to antigens
on the cell surface are not taken up into cells. Some receptors are taken
up into cells (endocytosis) after binding to a ligand, which phenomenon
is called internalization. Similarly, internalization may also occur for
antibodies bound to cell surface antigens. When internalization occurs,
the Fc region responsible for most effector functions caused by the
antibody is also taken up into the cell, which as a result inhibits the
binding to the Fc region of effector cells or complements that are
outside the antigen-expressing cells, and finally the antibody effector
function. Therefore, when selecting an antibody with effector function,
it is important to choose antigens that cause less antibody
internalization.

[0049]Further, to treat cancer by using antibodies, it is also important
to select antigens which expression level is low in normal organs to
avoid side effects.

[0050]Thus, the present inventors identified a number of genes with
specifically enhanced expression in the cancer cells but showing low
expression levels in normal cells through gene expression analysis with
cDNA microarrays of lung cancer cells and normal cells collected from
lung cancer patients. Among these genes, those showing low expression
levels in major organs were selected as candidate target genes for lung
cancer therapy.

[0051]The candidate target genes included the desmocollin 2 (DSC2) gene.
The amino acid sequence coded by the DSC2 gene is expected to comprise a
signal peptide at its N-terminus, and thus was expected to be a protein
expressed on the surface of the cytoplasmic membrane. Through a forced
expression system, localized expression of c-myc-His-tagged DSC2 on the
cytoplasmic membrane was confirmed via Immuno-fluorescence microscopy,
and DSC2 was thought to be a transmembrane protein. Thus, the present
inventors expected that DSC2 serves as a useful clinical marker and
therapeutic target for lung cancer.

[0052]Specifically, antibodies against proteins encoded by these candidate
target genes were examined for their ability of effector function to
induce potent cytotoxicity and to finally damage lung cancer cells. As a
result, anti-DSC2 antibodies could be confirmed to have effector function
against DSC2-expressing cells. Furthermore, these antibodies were
confirmed to cause similar effects on other cancer cell lines, such as
colon, pancreatic, prostate, breast, gastric, and liver cancer cell lines
wherein DSC2 was over-expressed. According to these discoveries, the
inventors contemplated that antibodies against DSC2 can be used for
cancer therapy with little danger of side effects.

I-1. Polypeptides

[0053]According to an aspect of the present invention, polypeptides having
any of the following amino acid sequences are provided:

[0054]Each of the above-mentioned amino acid sequences are the sequence
determined for the complementarity determining regions (CDR) of mouse
antibodies which regions were used for constructing chimeric antibodies
or human antibodies that were confirmed to be effective to damage cells
expressing DSC2. It is known that a CDR region alone even weak can
recognize and specifically bind to its antigen. Further, it is generally
known that the role of CDR3 among the three CDRs is particularly high in
the binding of the antibody to its antigen. Thus, in some cases, the
above-mentioned polypeptides of the present invention may be used alone
in the diagnosis or treatment of diseases wherein the expression of DSC2
is involved.

[0055]CDR graft technology is known in the art ("Immunoglobulin genes",
Academic Press (London), pp 260-74, 1989; Michael A et al., Proc Natl
Acad Sci USA 91: 969-73, 1994). According to this technology, the CDRs of
an antibody are replaced with the CDRs of another antibody. Through such
replacement, the binding specificity of the former antibody is changed to
that of the latter antibody. Among such chimeric antibodies, those whose
framework amino acids are derived from a human antibody are called
humanized antibodies, and are expected to cause less side effects when
used in cancer therapy for human. Thus, the present polypeptide
consisting of any of the aforementioned CDR amino acid sequences can also
be used for preparing such chimeric antibody.

[0056]When the present polypeptide is used for preparing a chimeric
antibody, it is preferable to use them in combination as follows:

[0057]Polypeptides which comprise the amino acid sequence in a combination
as above, wherein the CDR1, CDR2, and CDR3 are separated by framework
amino acid sequences are also encompassed by the present polypeptide.
Furthermore, the present polypeptides may comprise both the VH and VL
regions. Namely, a polypeptide of the present invention may comprise all
the amino acid sequences of pairs of aforementioned groups 1-1 and 1-2,
groups 2-1 and 2-2, groups 3-1 and 3-2, or groups 4-1 and 4-2 separated
by appropriate amino acid sequences (e.g., framework amino acid sequence,
etc.) to retain the specific binding ability of the original antibodies.

[0058]According to another aspect, the present invention provides
polypeptides having any of the amino acid sequences selected from the
group of:

[0059]Each of the above-mentioned amino acid sequences are the sequence
determined for the variable regions of either the light chain or the
heavy chain of mouse or human antibodies that were confirmed to be
effective to damage cells expressing DSC2.

[0060]Various chimeric antibodies are known in the art. For example, the
Fc region of an antibody may be linked with arbitrary variable regions.
The Fc region of IgA, IgE, or IgG class antibody is essential for
generating ADCC. Similarly, the Fc region of IgM or IgG class antibody is
required for generating CDC. Therefore, for utilization in cancer therapy
on human, the antibody preferably has the Fc region of a human antibody
to achieve the generation of ADCC and/or CDC. Thus, a polypeptide
consisting of any of the aforementioned VH and VL amino acid sequences
can be used for preparing such chimeric antibody wherein the Fc region of
a human antibody is linked to the variable region that had been confirmed
to be effective to damage cells expressing DSC2 by the present inventors.
Since the IgG1 class antibodies triggers both ADCC and CDC, the Fc region
of an IgG1 class antibody is preferable for the present invention. Much
preferred may be the Fc region of a human IgG1 antibody.

[0061]Generally, it is known that modifications of one or more amino acid
in a protein do not influence the function of the protein. One of skill
in the art will recognize that individual additions, deletions,
insertions, or substitutions to an amino acid sequence which alters a
single amino acid or a small percentage of amino acids is a "conservative
modification" wherein the alteration of a protein results in a protein
with similar functions. Conservative substitution tables providing
functionally similar amino acids are well known in the art. For example,
the following eight groups each contain amino acids that are conservative
substitutions for one another:

[0070]Such conservatively modified polypeptides are included in the
present polypeptides. However, proteins applicable for the method are not
restricted thereto and may include non-conservative modifications so long
as they retain the specific binding ability to DSC2.

[0071]In addition to the above-mentioned modification, the present
polypeptides may be further linked to other substances so long as they
retain their specific binding ability. Usable such other substances
include: peptides, lipids, sugar and sugar chains, acetyl groups, natural
and synthetic polymers, etc. These kinds of modifications may be
performed to confer additional functions or to stabilize the
polypeptides.

[0080]Alternatively, the polypeptides may be obtained adopting any known
genetic engineering methods for producing polypeptides (e.g., Morrison J,
J. Bacteriology 132: 349-51, 1977; Clark-Curtiss & Curtiss, Methods in
Enzymology (eds. Wu et al.) 101: 347-62, 1983). For example, first, a
suitable vector comprising a polynucleotide encoding the objective
protein in an expressible form (e.g., downstream of a regulatory sequence
comprising a promoter) is prepared, transformed into a suitable host
cell, and then the host cell is cultured to produce the protein. The
protein may also be produced in vitro adopting an in vitro translation
system.

I-2. Antibodies

[0081]As an aspect of the present invention, a polypeptide of the
invention may be an antibody. The antibody of the present invention may
belong to any class or subclass of immunoglobulins. Since the IgG1 class
antibodies triggers both ADCC and CDC, and non-specific binding of this
class of antibody is considered to be lowest among the immunoglobulin
classes and subclasses, an IgG1 class antibody is particularly preferable
for the present invention.

[0082]Further, when the antibodies are used for therapy for animals, it is
preferred to select an antibody derived from the same species or at
least, those having the Fc region or the constant region of an antibody
from the same species. Namely, when used for treating humans, it is
preferred to use human antibodies or humanized antibodies.

[0083]Further, the present invention includes monoclonal and polyclonal
antibodies, modified antibodies such as chimeric antibodies (humanized
antibodies, scFv, etc.) that retain the specific antigen-binding ability
of the original antibody, and antibody fragments (e.g., Fv, Fab,
F(ab')2, etc.) so long as they retain their specific binding ability
to its antigen. However, the present invention is not restricted to any
of the aforementioned antibodies.

[0084]As an embodiment, the present antibody includes the following
sequences as the CDRs:

[0085]Preferably, the CDR1, CDR2, and CDR3 sequences therein are separated
by appropriate framework amino acid sequences. More preferably, the
antibody of the present invention has the VH and VL sequences selected
from the group of:

[0086]Such preferred examples of antibodies may be, for example, 48-5,
s10-4, ch48-5, chs10-4, 332, and 545, all of which were prepared in the
Example; but the present invention is not restricted thereto.

[0087]It is particularly preferred that the antibody of the present
invention generates effector function. Thus, the present invention
further relates to antibodies against DSC2 that show at least one
effector function. Suitable antibodies of the invention show effector
function such as ADCC, CDC, or both. Antibodies comprising the Fc region
of IgA, IgE, or IgG are essential for expressing ADCC. Equally, the
antibody Fc region of IgM or IgG is preferable for expressing CDC.
However, the antibodies of the present invention are not limited so long
as they drive a desired effector function.

[0088]Variants, analogs or derivatives of the Fc portion may be
constructed by, for example, making various substitutions of residues or
sequences, and may be used for the present antibody. Variant (or analog)
polypeptides include insertion variants, wherein one or more amino acid
residues supplement an Fc amino acid sequence. Insertions may be located
at either or both termini of the protein, or may be positioned within
internal regions of the Fc amino acid sequence. Insertional variants with
additional residues at either or both termini can include, for example,
fusion proteins and proteins including amino acid tags or labels. For
example, the Fc molecule may optionally contain an N-terminal Met,
especially when the molecule is to be expressed recombinantly in a
bacterial cell such as E. coli.

[0089]In Fc deletion variants, one or more amino acid residues are removed
in the Fc. Deletions can be included at one or both termini of the Fc
polypeptide, or with removal of one or more residues within the Fc amino
acid sequence. Deletion variants, therefore, include all kind of
fragments of an Fc polypeptide sequence.

[0090]In Fc substitution variants, one or more amino acid residues of an
Fc polypeptide are removed and replaced with alternative residues. In one
aspect, the substitutions are conservative in nature, however, the
invention embraces substitutions that are non-conservative.

[0091]Preferably, the parent polypeptide Fc region used in the present
antibody having effector function is a human Fc region, e.g., native
human Fc region like those from human IgG1 (A and non-A allotypes)
or human IgG3. In one embodiment, the variant with improved ADCC
mediates ADCC substantially more effectively than an antibody with a
native sequence IgG1 or IgG3 Fc region and the antigen-binding
region of the variant. Preferably, the variant comprises, or consists
essentially of, substitutions of two or three of the residues at
positions 298, 333 and 334 of the Fc region. The numbering of the
residues in an immunoglobulin heavy chain is that of the EU index as in
Kabat et al., (Sequences of Proteins of Immunological Interest, Fifth
Edition, National Institute of Health, Bethesda, Md., 1991), expressly
incorporated herein by reference. Most preferably, residues at positions
298, 333 and 334 are substituted, (e.g., with alanine residues).
Moreover, in order to generate the Fc region variant with improved ADCC
activity, one will generally engineer an Fc region variant with improved
binding affinity for FcγRIII, which is thought to be an important
FcR for mediating ADCC. For example, one may introduce an amino acid
modification (e.g., an insertion, a deletion, or a substitution) into the
parent Fc region at any one or more of amino acid positions 256, 290,
298, 312, 326, 330, 333, 334, 360, 378 or 430 to generate such a variant.
The variant with improved binding affinity for FcγRIII may further
have reduced binding affinity for FcγRII, especially reduced
affinity for the inhibiting FcγRIIB receptor.

[0092]In any event, any variant amino acid insertions, deletions and/or
substitutions (e.g., from 1-50 amino acids, preferably, from 1-25 amino
acids, more preferably, from 1-10 amino acids) are contemplated and are
within the scope of the present invention. Conservative amino acid
substitutions will generally be preferred. Furthermore, alterations may
be in the form of altered amino acids, such as peptidomimetics or D-amino
acids, as already explained for the present polypeptides including
antibodies.

[0093]DSC2 or a fragment thereof can be used as mimunogen to obtain an
antibody of the present invention. DSC2 can be derived from any species,
preferably from a mammal such as a human, mouse, or rat, and more
preferably from a human through conventional purification techniques.
Moreover, the nucleotide and amino acid sequences of human DSC2 are known
(cDNA nucleotide sequence of DSC2 type 2b (GenBank Accession No.
NM--004949; SEQ ID NO: 1) and DSC2 type 2a (GenBank Accession No.
NM--024422; SEQ ID NO:2), and the corresponding amino acid sequences
are described in SEQ ID NOs: 3 (GenBank Accession No. NP--004940)
and 4 (GenBank Accession No. NP--077740), respectively). Thus, to
obtain an immunogen for preparing the present DSC2 antibody, a person may
chemically synthesize or genetically produce DSC2 or antigenic fragments
thereof based on these sequence information. For example, one skilled in
the art can routinely isolate or construct a polynucleotide comprising
the objective nucleotide sequence, insert the gene into a suitable
expression vector to transform a suitable host cell, and obtain a protein
comprising the target amino acid sequence by culturing the host cell
under suitable conditions for expression of the protein from the cells or
the culture supernatant. Furthermore, cells expressing the DSC2 protein
or a fragment thereof can themselves be used as immunogens.

[0094]When using a fragment of DSC2 as the immunogen, it is particularly
preferable to select an amino acid sequence which comprises a region
predicted to be an extra-cellular domain. The region of positions 1 to 32
of the N-terminus of DSC2 is predicted to correspond to a signal sequence
(Greenwood M D et al., Genomics 44: 330-5, 1997.). Thus, it is preferred
to avoid the use of this region as an immunogen. Further, it is preferred
to adopt the extra-cellular domains of the DSC2 as the immunogen to
obtain an antibody of the present invention.

[0095]Methods for immunizing animals with antigens are well known in the
art, and include intraperitoneal and subcutaneous antigen injections.
Specifically, antigens can be diluted and suspended in an appropriate
amount of phosphate buffered saline (PBS), physiological saline, or the
like. As desired, antigen suspensions can be mixed with an appropriate
amount of standard adjuvant such as Freund's complete adjuvant, and
administered to mammals after emulsification. Subsequently, it is
preferable that antigens mixed with an appropriate amount of Freund's
incomplete adjuvant are administered in multiple doses every four to 21
days. An appropriate carrier can also be used for immunization. After
carrying out immunization as outlined above, the antibody level in the
serum of the immunized animal may be examined through standard methods.

[0096]Polyclonal antibodies against the DSC2 protein can be prepared from
the immunized mammal for which an increase in the level of desired
antibody could be confirmed. This can be achieved by collecting blood or
serum from these animals. The polyclonal antibody of the present
invention may be the collected serum itself or may be purified from the
serum. For example, chromatography using affinity columns equipped with
DSC2 protein or antigenic fragments thereof may be used for such
purification. Furthermore, IgG and IgM can be prepared by further
purification using protein A or protein G column.

[0097]To prepare monoclonal antibodies, first, antibody-forming cells are
collected from mammals immunized with immunogens and that have been
confirmed to show increased level of the desired antibody in serum. The
cells are preferably collected from the spleen. The collected
antibody-forming cells are fused with preferable parent cells, for
example, mammalian myeloma cells, and more preferably, myeloma cells that
have acquired properties for selection of fusion cells by pharmaceutical
agents. The fusion can be achieved through any known methods, for example
the methods of Milstein et al. (Galfre G and Milstein C, Methods Enzymol
73: 3-46, 1981).

[0098]Then, the hybridomas produced by cell fusion may be selected by
culturing in a standard selective medium such as HAT medium (medium
comprising hypoxanthine, aminopterin, and thymidine). Cell culture in HAT
medium is usually continued for several days to several weeks, a period
sufficient enough to kill all cells other than the desired hybridomas
(unfused cells). Standard limiting dilutions are then carried out, and
hybridoma cells that produce the desired antibodies are screened and
cloned.

[0102]Any mammal can be immunized with the immunogen for the production of
the present antibody. However, when preparing a monoclonal antibody by
producing a hybridoma, it is preferable to consider compatibility with
the parent cell used in the cell fusion for producing the hybridoma.

[0104]Furthermore, the use of transgenic animals comprising a repertoire
of human antibody genes is also known in the art (Ishida I, et al.,
Cloning and Stem Cells 4: 91-102, 2002). Similarly with other animals, to
obtain a human monoclonal antibody, the transgenic animals are immunized,
antibody-producing cells are then recovered from the animals, fused with
myeloma cells to yield hybridomas, and anti-protein human antibodies can
be prepared from these hybridomas (see International Publications Nos.
92-03918, 94-02602, 94-25585, 96-33735, and 96-34096).

[0105]Alternatively, lymphocytes that are immortalized with cancer genes
can be used for monoclonal antibody production. For example, human
lymphocytes infected with EB virus or the like, can be immunized in vitro
with immunogens. The immunized lymphocytes are then fused with
human-derived myeloma cells able to divide unlimitedly (U266, etc.), thus
obtaining hybridomas that produce the desired human antibodies (Japanese
Patent Application Kokai Publication No. (JP-A) Sho 63-17688).

[0106]Once a monoclonal antibody has been obtained via any of the
above-mentioned methods, it also can be prepared using genetic
engineering methods (e.g., see Borrebaeck C A K and Larrick J W,
Therapeutic Monoclonal Antibodies, MacMillan Publishers, UK, 1990). For
example, a recombinant antibody can be prepared by cloning the DNA that
encodes an objective antibody from the antigen-producing cell, such as
hybridoma or immunized lymphocyte that produce the antibody; then
inserting the cloned DNA into an appropriate vector; and transforming the
vector into a suitable host cell. Such recombinant antibodies are also
encompassed by the present invention.

[0107]Modified antibodies are also included in the present invention. Such
modified antibodies can be obtained, for example, by chemical
modification. For example, an antibody can be modified by linking to a
molecule, such as polyethylene glycols (PEGs). Such chemical modification
methods for antibodies are conventional to those skilled in the art and
any known method may be adopted in the present invention. The antibodies
can also be modified by other proteins. For example, an antibody linked
with another protein molecule may be produced through genetic
engineering. That is, a fusion protein of the antibody and the other
protein can be expressed from an expression vector which includes a gene
wherein the antibody gene and the gene coding for the other protein are
linked. As a preferred example of the present invention, to enhance the
effector function of the antibody, it may be linked with a cytokine or
chemokine. It has been reported that the antibody effector function is
enhanced via the linkage with IL-2, GM-CSF, or the like (Human Antibody
10: 43-9, 2000). IL-2, IL-12, GM-CSF, TNF, eosinophil chemotactic
substance (RANTES) and the like can be used in the present invention to
enhance the effector function of an antibody.

[0108]Moreover, modified antibodies include chimeric antibodies which, for
example, are represented by humanized antibodies, wherein a variable
region derived from a non-human antibody is conjugated to the constant
region of a human antibody, or wherein CDRs from a non-human antibody is
fused with the framework region (FR) derived from a human antibody
(CDR-grafted antibody). Such chimeric antibodies may be obtained via
standard techniques of molecular biology (see, e.g., Jones et al., Nature
321:522-5, 1986; Riechmann et al., Nature 332:323-7, 1988; and Presta,
Curr Opin Struct Biol 2:593-6, 1992) for the production of humanized
antibodies.

[0109]For example, first, genes encoding the variable region or CDR of an
antibody of interest are prepared by polymerase chain reaction (PCR) or
the like from RNA of antibody-producing cells (see, e.g., Larrick et al.,
"Methods: a Companion to Methods in Enzymology", Vol. 2: 106, 1991;
Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies" in
Monoclonal Antibodies: Production, Engineering and Clinical Application;
Ritter et al. (eds.), page 166, Cambridge University Press, 1995, and
Ward et al., "Genetic Manipulation and Expression of Antibodies" in
Monoclonal Antibodies: Principles and Applications; Birch et al. (eds.),
page 137, Wiley-Liss, Inc., 1995). The prepared variable region-encoding
genes are linked with genes that code for the constant region or
framework regions. The genes encoding the constant region or framework
regions may be determined similarly to the CDR-encoding genes, or it is
also possible to prepare them based on sequence information of
pre-existing antibodies. DNA sequences coding for the chimeric and
CDR-grafted products may be synthesised completely or in part using
oligonucleotide synthesis techniques. For example, oligonucleotide
directed synthesis as described by Jones et al. (Nature 321:522-5, 1986)
may be used. Further, in some cases, site-directed mutagenesis and
polymerase chain reaction techniques may be used as appropriate.
Techniques for oligonucleotide directed mutagenesis of a pre-existing
variable region described by Verhoeyen et al. (Science 239: 1534-6, 1988)
or Riechmann et al. (Nature 332: 323-7, 1988) may be employed for
modifying the sequence of the variable region to, for example, enhance
the binding ability of the chimeric antibody. In addition, if needed,
enzymatic filling in of gapped oligonucleotides using T4 DNA polymerase
as, for example, described by Queen et al., (Proc Natl Acad Sci USA 86:
10029-33, 1989; WO 90/07861) may be used.

[0110]In addition, the present antibodies encompass those wherein one or
more amino acids have been replaced with other amino acids, or those
wherein one or more amino acids are deleted, or added (including
insertion) so long as the resulting antibody retains the binding ability
to DSC2. Conventional methods used for other polypeptide, such as
site-directed mutagenesis, may be employed for obtaining this kind of
modified antibodies.

[0111]Similarly, fragments of any of the aforementioned present antibodies
are also encompassed by the present invention so long as the resulting
fragment retains the binding ability to DSC2. Such fragments are
represented by Fv, Fab, and F(ab')2, that can be obtained by
treating antibodies with appropriate enzymes, such as papain or pepsin.
However, much smaller fragments of the variable region of an antibody is
included in the present invention. The fragments can be also obtained
through chemical synthesis or conventional gene engineering methods by
constructing genes encoding the fragments and expressing them.

[0112]Single-chain Fv (scFv) is also included in the present antibody. An
sFv comprises the VH and VL domains of an antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the scFv to form the desired structure
for antigen binding. A number of methods have been described to discern
chemical structures for converting the naturally aggregated but
chemically separated light and heavy polypeptide chains from an antibody
V region into an scFv molecule which will fold into a three dimensional
structure substantially similar to the structure of an antigen-binding
site (U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,946,778; Pluckthun in
The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore
eds., Springer-Verlag, New York, pp. 269-315, 1994) which all can be
employed in the present invention.

[0113]Any suitable host cell/vector system may be used for expression of
the DNA sequences coding for the modified antibodies. Bacterial, e.g., E.
coli, and other microbial systems may be used, in particular for
expression of antibody fragments such as FAb and (Fab')2 fragments,
and especially Fv fragments and single-chain antibody fragments, e.g., sc
Fvs. Eucaryotic, e.g., mammalian, host cell expression systems may be
used, in particular, for production of larger CDR-grafted antibody
products, including complete antibody molecules. Suitable mammalian host
cells include CHO cells and myeloma or hybridoma cell lines.

[0114]The antigen-binding ability of an antibody of the present invention
can be measured by using absorbance measurements, enzyme linked
immunosorbent assays (ELISA), enzyme immunoassays (EIA),
radioimmunoassays (RIA) and/or immunofluorescence methods. In ELISA, the
antibody is immobilized on a plate, and an antigen thereto (e.g., the
whole DSC2 protein or a fragment thereof) is added to the plate, and then
a sample comprising the desired antibody such as the culture supernatant
of cells that produce the antibody or purified antibody is added. A
secondary antibody that recognizes the primary antibody and has been
tagged with an enzyme such as alkaline phosphatase is then added, and the
plate is incubated. After washing, an enzyme substrate such as
p-nitrophenyl phosphate is added to the plate, absorbance is measured,
and the antigen-binding ability of the objective sample is evaluated. The
evaluation may be achieved using BIAcore (Pharmacia).

[0115]In addition, the effector function of the antibodies may be
examined, for example, to select monoclonal antibodies which comprise
more powerful effector function. For example, to assess ADCC activity of
a molecule of interest, an in vitro ADCC assay, such as that described in
U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Alternatively,
ADCC activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. (Proc Natl Acad
Sci USA 95: 652-56, 1998). In addition, by following the methods outlined
in the Example, the antibody effector function can also be evaluated. For
example, target DSC2-expressing cells are incubated with effector cells
in the presence of an antibody whose effector function is to be
evaluated. If target cell destruction is detected, the antibody can be
confirmed to have effector function that induces ADCC. The level of
observed target cell destruction, in the absence of either antibodies or
effector cells, can be compared as a control. Cells expressing DSC2 can
be used as the target cells, including the variety of cell lines
confirmed to express DSC2 in the Examples. These cell lines can be
obtained from cell banks.

[0116]Further, to assess CDC activity of a molecule of interest, a CDC
assay, e.g., as described by Gazzano-Santoro et al. (J Immunol Methods
202: 163-71, 1997) may be performed.

[0117]The antibodies of the present invention can be used not only for
purifying or detecting DSC2, but also serve as candidates for agonists
and antagonists of this protein. These antibodies can also be applied to
antibody therapies for diseases wherein the expression of DSC2 is
implicated. When using for treating humans, human antibodies or humanized
antibodies are preferred due to their low immunogenicity.

II. Polynucleotides

[0118]Furthermore, the present invention provides polynucleotides encoding
the above-described polypeptides of the present invention, including the
antibodies. Any form of the polynucleotide of the present invention can
be used so long as it encodes the present polypeptides, including mRNA,
RNA, cDNA, genomic DNA, chemically synthesized polynucleotides, and the
like. The present polynucleotides include those comprising a given
nucleotide sequences as well as their degenerate sequences, so long as
the resulting substance encodes the objective polypeptide of the present
invention or equivalents thereof.

[0119]Preferably, the polynucleotide of the present invention includes a
sequence coding for a polypeptide which consists of or comprises the
amino acid sequence selected from the group of:

[0120]The present polynucleotide may encode an antibody. Such antibody
encoding polynucleotide of the present invention may encode an antibody
that comprises a complementarity determining region (CDR) having
sequences as follows:

[0121]Preferably, the above-described CDR1, CDR2, and CDR3 sequences are
separated by appropriate framework amino acid sequences so that the
resulting fragment shows binding affinity to DSC2.

[0122]In addition, a polynucleotide of the present invention may encode an
antibody that comprises the mouse VH amino acid sequence of SEQ ID NO: 20
or 22, and mouse VL amino acid sequence of SEQ ID NO; 21 or 23, or an
antibody that comprises the human VH amino acid sequence of SEQ ID NO: 16
or 18, and the human VL amino acid sequence of SEQ ID NO: 17 or 19.

[0123]Furthermore, the present polynucleotide encoding an antibody may
comprise a region that encodes for an Fc region of the antibody.
Preferable Fc region encoded by the polynucleotide includes that of human
IgG1, but the present invention is not restricted thereto.

[0124]The polynucleotide of the present invention can be prepared by
methods known to those skilled in the art including genetic engineering
methods and chemical synthesis. For example, it can be prepared by:
preparing a cDNA library from cells which express the objective protein
(e.g., antibody) of the invention, and conducting hybridization using a
known partial sequence of the objective protein (e.g., if the objective
protein is an antibody, the sequence coding for the constant region or
framework region) as a probe. cDNA library construction can be achieved,
for example, by the method described in Sambrook et al. (Molecular
Cloning, Cold Spring Harbor Laboratory Press, 1989) or commercially
available cDNA libraries may be used. Such library can also be prepared
by extracting RNAs from cells expressing the objective protein,
synthesizing oligo DNAs based on the known sequence of the objective
protein, conducting PCR using the oligo DNAs as primers, and amplifying
cDNAs encoding the objective protein.

[0125]In addition, by sequencing the nucleotide sequence of the obtained
cDNA, the translation region encoded by the cDNA can be routinely
determined, and the amino acid sequence of the objective protein can also
be easily deduced. Moreover, the genomic DNA library can also be screened
for the present protein using similar probes as screening the cDNA
library to isolate genomic DNA of the objective protein.

[0126]More specifically, mRNAs may first be prepared from a cell, tissue,
or organ in which the objective protein is expressed. Known methods can
be used to isolate mRNAs, for instance, total RNA may be prepared by
guanidine ultracentrifugation (Chirgwin et al, Biochemistry 18: 5294-9,
1979) or AGPC method (Chomczynski and Sacchi, Anal Biochem 162: 156-9,
1987). In addition, mRNA may be purified from total RNA using mRNA
Purification Kit (Pharmacia) and the like, or may be directly purified by
QuickPrep mRNA Purification Kit (Pharmacia).

[0128]A desired DNA fragment is prepared from the PCR products and ligated
with a vector DNA. The recombinant vectors are used to transform E. coli
and the like, and a desired recombinant vector is prepared from a
selected colony. The nucleotide sequence of the desired DNA can be
verified by conventional methods including dideoxynucleotide chain
termination.

[0129]The nucleotide sequence of a polynucleotide of the invention may be
designed to be expressed more efficiently by taking into account the
frequency of codon usage in the host used for the expression (Grantham et
al., Nucleic Acids Res 9: 43-74, 1981). The sequence of the
polynucleotide of the present invention may be altered by commercially
available kits or conventional methods. For instance, the sequence may be
altered by digestion with restriction enzymes, insertion of synthetic
oligonucleotides or appropriate polynucleotide fragments, addition of
linkers, and/or insertion of an initiation codon (ATG) and/or a stop
codon (TAA, TGA, or TAG).

[0130]The present polynucleotide may be used for preparing a polypeptide
of the invention. Furthermore, it may also be used for diagnosis and gene
therapy against various diseases where DSC2 expressing cells are
involved.

III. Vectors and Host Cells

[0131]The present invention also provides a vector into which the above
polynucleotide of the present invention has been inserted. A vector of
the invention is useful to keep a polynucleotide, especially a DNA, of
the present invention in host cell, to express the polypeptide of the
present invention, or to administer the polynucleotide of the present
invention for gene therapy.

[0132]When E. coli is used a host cell and the vector is amplified and
produced in a large amount in E. coli (e.g., JM109, DH5alpha, HB101,
XLlBlue, etc.) the vector should have "ori" to be amplified in E. coli
and a marker gene for selecting transformed E. coli (e.g., a
drug-resistance gene selected by a drug such as ampicillin, tetracycline,
kanamycin, chloramphenicol, etc.). For example, M13-series vectors,
pUC-series vectors, pBR322, pBluescript, pCR-script, and the like can be
used. In addition, pGEM-T, pDIRECT, and pT7 can also be used for
subcloning and extracting cDNA as well as the vectors described above.

[0133]When, a vector is used to produce the polypeptide of the present
invention, an expression vector is especially useful. For example, an
expression vector to be expressed in E. coli should have the above
characteristics to be amplified in E. coli. When E. coli, such as JM109,
DHSalpha, HB101, or XLlBlue, are used as a host cell, the vector should
have a promoter or the like, for example, lacZ promoter (Ward et al.,
Nature 341: 544-6, 1989; FASEB J 6: 2422-7, 1992), araB promoter (Better
et al., Science 240: 1041-3, 1988) or T7 promoter or the like, that can
efficiently express the desired gene in E. coli. In that respect,
pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP and pET (in
this case, the host is preferably BL21 which expresses T7 RNA
polymerase), for example, can be used instead of the above vectors.
Additionally, the vector may also contain a signal sequence for protein
secretion. An exemplary signal sequence that directs the protein to be
secreted to the periplasm of the E. coli is the pelB signal sequence (Lei
et al., J Bacteriol 169: 4379-83, 1987). Means for introducing the
vectors into the target host cells include, for example, the calcium
chloride method and the electroporation method.

[0135]In order to express the vector in animal cells, such as CHO, COS, or
NIH3T3 cells, the vector should have a promoter necessary for expression
in such cells, for example, the SV40 promoter (Mulligan et al., Nucleic
Acids Res 277: 108, 1979), the MMLV-LTR promoter, the EFlalpha promoter
(Mizushima et al., Nucleic Acids Res 19: 5322, 1990), the CMV promoter,
and the like, and preferably a marker gene for selecting transformants
(e.g., a drug resistance gene selected by a drug (neomycin, G418, etc.)).
Examples of known vector with these characteristics include, for example,
pMAM, pDR2, PBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0136]As has been mentioned above, when the polypeptide to be expressed is
an antibody fragment, such as FAb and (Fab')2 fragments, or sc Fvs,
bacterial, e.g., E. coli, and other microbial systems are suitably used.
Alternatively, eucaryotic, e.g., mammalian, host cell expression systems
may be used, in particular, for production of larger polyeptides of the
present invention like CDR-grafted antibody products, and complete
antibody molecules. Suitable mammalian host cells include CHO cells and
myeloma or hybridoma cell lines.

[0138]The pharmaceutical composition can be used to treat any pathological
condition associated with the expression of DSC2. In typical embodiments,
the cell damaged by the present pharmaceutical composition is a cancer
cell, such as lung, colon, pancreatic, prostate, breast, gastric, or
liver cancer cell. More specifically, non-small cell lung cancer (NSCLC),
colorectal carcinoma, pancreatic carcinoma, prostate carcinoma, breast
duct carcinoma, tubular adenocarcinoma of the stomach, hepatocellular
carcinoma (HCC) may be treated using the present compositions.

[0139]Any of the natural antibodies and modified antibodies described
above under the item of "I-2. Antibodies" may be adopted for the present
pharmaceutical composition so long as they show antibody effector
function. It is preferred to use an isolated or purified antibody for the
present composition. The antibody contained in the present pharmaceutical
composition typically is a monoclonal antibody. However, the present
invention is not limited thereto and any antibodies may be used for the
present pharmaceutical composition so long as they comprise a desired
effector function. Preferred effector functions include ADCC, CDC, and
both. For example, antibodies comprising the Fc region of IgA, IgE, or
IgG are essential for expressing ADCC. Similarly, the antibody Fc region
of IgM or IgG is preferable for expressing CDC. Particularly preferred
antibodies included in the composition are those belonging to the
immunoglobulin class of IgG1. When the composition is used for treating
human, human-derived antibodies belonging to these classes are
particularly preferable in the present invention.

[0140]Furthermore, the antibody included in the present pharmaceutical
composition may, in some embodiments, linked to cytotoxic agents via well
known techniques. Numerous cytotoxic agents are known in the art and
those that can be used in the present invention include, but are not
limited to, cytotoxic drugs, toxins, and active fragments of such toxins.
Suitable toxins and their corresponding fragments include diphtheria, A
chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin,
phenomycin, enomycin, auristatin and the like. Cytotoxic agents also
include radiochemicals made by conjugating radioisotopes to the antibody
or binding of a radionuclide to a chelating agent that has been
covalently attached to the antibody. Methods for preparing such
conjugates are well known in the art.

[0141]The present pharmaceutical composition can be administered to humans
or other animals. In the present invention, animals other than humans to
which the composition can be administered include mice, rats, guinea
pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons,
and chimpanzees.

[0142]The composition is formulated into a dosage form using known
pharmaceutical formulation methods. For example, depending on
requirements, it can be prepared to an injectable form that can be
parenterally administered by making it as a sterile solution or
suspension with water or other arbitrary pharmaceutically acceptable
fluid. For example, the antibody to be included in the pharmaceutical
composition can be mixed with acceptable carriers or solvents,
specifically sterile water, physiological saline, vegetable oils,
emulsifiers, suspension agents, surfactants, stabilizers, flavoring
agents, excipients, solvents, preservatives, binding agents and the like,
into a generally accepted unit dosage essential for use as a
pharmaceutical agent. The phrase "pharmaceutically acceptable" indicates
that the substance is inert and includes conventional substances used as
diluent or vehicle for a drug. Suitable excipients and their formulations
are described, for example, in Remington's Pharmaceutical Sciences,
16th ed. (1980) Mack Publishing Co., ed. Oslo et al.

[0143]Other isotonic solutions comprising physiological saline, glucose,
and adjuvants (such as D-sorbitol, D-mannose, D-mannitol, and sodium
chloride) can be used as the injectable aqueous solution. They can also
be used with appropriate solubilizers such as alcohols, specifically
ethanols and polyalcohols (for example, propylene glycols and
polyethylene glycol), and non-ionic surfactants (for example polysorbate
80® or HCO-50).

[0144]Sesame oils or soybean oils can be used as an oleaginous solution,
and benzyl benzoate or benzyl alcohols can be used with them as a
solubilizer. Buffer solutions (phosphate buffers, sodium acetate buffers,
etc.), analgesics (procaine hydrochloride, etc.), stabilizers (benzyl
alcohol, phenols, etc.), and antioxidants can be used in the formulation.
The prepared injections can be packaged into appropriate ampules.

[0145]Alternatively, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat
or prevent diseases associated with DSC2-expressing cells, such as
pancreatic, lung, colon, prostate, breast, gastric, and liver cancer, by
way of gene therapy. Gene therapy refers to therapy performed by the
administration to a subject of an expressed or expressible nucleic acid.
In this embodiment of the invention, the nucleic acids produce their
encoded antibody or antibody fragment that mediates a prophylactic or
therapeutic effect.

[0146]Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described
below.

[0148]In a preferred aspect, a composition of the invention comprises
nucleic acids encoding an antibody, said nucleic acids being part of an
expression vector that expresses the antibody in a suitable host. In
particular, such nucleic acids have promoters, preferably heterologous
promoters, operably linked to the antibody coding region, said promoter
being inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in which
the antibody coding sequences and any other desired sequences are flanked
by regions that promote homologous recombination at a desired site in the
genome, thus providing for intrachromosomal expression of the antibody
encoding nucleic acids (Koller and Smithies, Proc Natl Acad Sci USA
86:8932-5, 1989; Zijlstra et al., Nature 342:435-8, 1989). In specific
embodiments, the expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences encoding both
the heavy and light chains, or fragments thereof, of the antibody.

[0149]Delivery of the nucleic acids into a subject may be either direct,
in which case the subject is directly exposed to the nucleic acid or
nucleic acid-carrying vectors, or indirect, in which case, cells are
first transformed with the nucleic acids in vitro, then transplanted into
the subject. These two approaches are known, respectively, as in vivo or
ex vivo gene therapy.

[0150]In a specific embodiment, the nucleic acid sequences are directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known in the
art, e.g., by constructing them as part of an appropriate nucleic acid
expression vector and administering it so that they become intracellular,
e.g., by infection using defective or attenuated retrovirals or other
viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of
naked DNA, or by use of microparticle bombardment (e.g., a gene gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide which is
known to enter the nucleus, by administering it in linkage to a ligand
subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J Biol
Chem 262: 4429-32, 1987) (which can be used to target cell types
specifically expressing the receptors), etc. In another embodiment,
nucleic acid-ligand complexes can be formed in which the ligand comprises
a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid
to avoid lysosomal degradation. In yet another embodiment, the nucleic
acid can be targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO 92/06180,
WO 92/22635, WO92/20316, WO93/14188 or WO 93/20221). Alternatively, the
nucleic acid can be introduced intracellularly and incorporated within
host cell DNA for expression, by homologous recombination (Koller and
Smithies, Proc Natl Acad Sci USA 86:8932-5, 1989; Zijlstra et al., Nature
342:435-8, 1989).

[0151]In a specific embodiment, viral vectors that contains nucleic acid
sequences encoding an antibody of the invention are used. For example, a
retroviral vector can be used (see Miller et al., Methods Enzymol 217:
581-99, 1993). These retroviral vectors contain the components necessary
for the correct packaging of the viral genome and integration into the
host cell DNA. The nucleic acid sequences encoding the antibody to be
used in gene therapy are cloned into one or more vectors, which
facilitates delivery of the gene into a subject. More detail about
retroviral vectors can be found in Boesen et al., (Biotherapy 6: 291-302,
1994) which describes the use of a retroviral vector to deliver the mdr 1
gene to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J Clin Invest 93:
644-51, 1994; Kleim et al., Blood 83: 1467-73, 1994; Salmons and
Gunzberg, Hum Gene Ther 4: 129-41, 1993; Grossman and Wilson, Curr Opin
Genet Dev 3: 110-4, 1993.

[0154]Another approach to gene therapy involves transferring a gene to
cells in tissue culture by such methods as electroporation, lipofection,
calcium phosphate mediated transfection, or viral infection. Usually, the
method of transfer includes the transfer of a selectable marker to the
cells. The cells are then placed under selection to isolate those cells
that have taken up and are expressing the transferred gene. Those cells
are then delivered to a subject.

[0155]In this embodiment, the nucleic acid is introduced into a cell prior
to administration in vivo of the resulting recombinant cell. Such
introduction can be carried out by any method known in the art, including
but not limited to transfection, electroporation, microinjection,
infection with a viral or bacteriophage vector containing the nucleic
acid sequences, cell fusion, chromosome-mediated gene transfer,
microcellmediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign genes
into cells (see, e.g., Loeffler and Behr, Methods Enzymol 217: 599-618,
1993; Cotten et al., Methods Enzymol 217: 618-44, 1993; Cline M J,
Pharmacol Ther 29: 69-92, 1985) and may be used in accordance with the
present invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted. The
technique should provide for the stable transfer of the nucleic acid to
the cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.

[0156]The resulting recombinant cells can be delivered to a subject by
various methods known in the art. Recombinant blood cells (e.g.,
hematopoietic stem or progenitor cells) are preferably administered
intravenously. The amount of cells envisioned for use depends on the
desired effect, patient state, etc., and can be determined by one skilled
in the art.

[0157]Cells into which a nucleic acid can be introduced for purposes of
gene therapy encompass any desired, available cell type, and include but
are not limited to epithelial cells, endothelial cells, keratinocytes,
fibroblasts, muscle cells, hepatocytes; blood cells such as T
lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,
eosinophils, megakaryocytes, granulocytes; various stem or progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as
obtained from bone marrow, umbilical cord blood, peripheral blood, fetal
liver, etc. In a preferred embodiment, the cell used for gene therapy is
autologous to the subject.

[0158]In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced into
the cells such that they are expressible by the cells or their progeny,
and the recombinant cells are then administered in vivo for therapeutic
effect. In a specific embodiment, stem or progenitor cells are used. Any
stem and/or progenitor cells which can be isolated and maintained in
vitro can potentially be used in accordance with this embodiment of the
present invention (see e.g., WO 94/08598; Stemple and Anderson, Cell 71:
973-85, 1992; Rheinwald, Methods Cell Biol 21A: 229-54, 1980; Pittelkow
and Scott, Mayo Clin Proc 61: 771-7, 1986).

[0159]In a specific embodiment, the nucleic acid to be introduced for
purposes of gene therapy comprises an inducible promoter operably linked
to the coding region, such that expression of the nucleic acid is
controllable by controlling the presence or absence of the appropriate
inducer of transcription.

[0160]The pharmaceutical compositions comprise the active ingredients (a
polypeptide or polynucleotide of the present invention) at a
pharmaceutically effective amount. A "pharmaceutically effective amount"
of a compound is a quantity that is sufficient to treat and/or prevent
disorders wherein the expression of DSC2 plays important roles. An
example of a pharmaceutically effective amount may an amount that is
needed to decrease the number of DSC2 expressing cells in a cancerous
tissue when administered to a patient, so as to thereby treat or prevent
the disorders. The decrease may be, for example, at least a decrease of
about 5%, 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 99%, or 100%.
Alternatively, a pharmaceutically effective amount may be an amount that
leads to a decrease in size, prevalence, or metastatic potential of the
tumor in a subject.

[0161]The assessment to determine such a pharmaceutically effective amount
of an antibody of the present invention can be made using standard
clinical protocols including histopathologic diagnosis or through
identification of symptomatic anomalies such as chronic cough,
hoarseness, coughing up blood, weight loss, loss of appetite, shortness
of breath, wheezing, repeated bouts of bronchitis or pneumonia, and chest
pain.

[0162]The dose employed will depend upon a number of factors, including
the age and sex of the subject, the precise disorder being treated, and
its severity. Also the route of administration may vary depending upon
the condition and its severity. However, the determination of an
effective dose range for the identified compounds is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provide herein. The pharmaceutically or preventively
effective amount (dose) of a compound can be estimated initially from
cell culture assays and/or animal models.

[0163]If needed, a pharmaceutical composition comprising the antibody may
include any other therapeutic substance as an active ingredient so long
as the substance does not inhibit the in vivo effector function of the
antibody. It should be understood that in addition to the ingredients
particularly mentioned above, the formulations may include other agents
conventional in the art having regard to the type of formulation in
question.

[0164]In one embodiment of the present invention, a pharmaceutical
composition comprising the antibody may be included in articles of
manufacture and kits containing materials useful for treating the
pathological conditions of object. The article of manufacture may
comprise a container of any of the compounds with a label. Suitable
containers include bottles, vials, and test tubes. The containers may be
formed from a variety of materials, such as glass or plastic. The label
on the container should indicate the composition is used for treating or
preventing one or more conditions of the disease. The label may also
indicate directions for administration and so on.

[0165]In addition to the container described above, a kit comprising a
pharmaceutical composition comprising the antibody may optionally
comprise a second container housing a pharmaceutically-acceptable
diluent. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, syringes, and package inserts with instructions for
use.

[0166]The pharmaceutical compositions may, if desired, be presented in a
pack or dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise metal
or plastic foil, such as a blister pack. The pack or dispenser device may
be accompanied by instructions for administration.

V. Method for Damaging DSC2-Expressing Cells

[0167]The present invention also provides a method for damaging
DSC2-expressing cells. Specifically, the method comprises the step of
contacting the DSC2-expressing cells with anti-DSC2 antibodies. Through
the contact of the antibody, the cells are expected to be damaged due to
the effector function of the antibody.

[0169]Cells and antibodies can be contacted in vivo or in vitro. When
targeting in vivo cancer cells as the DSC2-expressing cells, the methods
of the present invention are in fact therapeutic methods or preventative
methods for cancers. Specifically, the present invention provides
therapeutic methods for cancers which comprise the following steps:
[0170]1) administering an antibody that binds DSC2 to a cancer patient,
and [0171]2) damaging cancer cells in the patient through the effector
function of the antibody that bound to those cells.

[0172]Any of the natural antibodies and modified antibodies described
above under the item of "I-2. Antibodies" may be employed for the present
method so long as they show antibody effector function. It is preferred
to use isolated or purified antibodies for the present method.
Alternatively, any of the pharmaceutical compositions described above
under the item of "IV. Pharmaceutical Compositions" may be adopted for
the present method.

[0174]In the present invention, the antibodies or pharmaceutical
compositions can be administered to patients, for example,
intraarterially, intravenously, or percutaneously, or intranasally,
transbronchially, locally, or intramuscularly. Intravascular
(intravenous) administration by drip or injection is an example of a
general method for systematic administration to lung, colon, pancreatic,
prostate, breast, gastric or liver cancer patients. Methods of locally
concentrating the administered agent to the primary focus or metastatic
focus in the lung include local injection using a bronchoscope
(bronchoscopy) and local injection under CT guidance or with
thoracoscopy. Methods of locally concentrating the agents to the primary
focus or metastatic focus in the liver include local injection using a
hepatic portal injection or arterial infusion. In addition, methods in
which an intraarterial catheter is inserted near a vein that supplies
nutrients to cancer cells to locally inject anti-cancer agents, are
effective as local control therapies for metastatic focuses as well as
primary focuses of lung, colon, pancreatic, prostate, breast, gastric or
liver cancer.

[0175]Although dosage and administration methods vary according to patient
body weight and age, and administration method, these can be routinely
selected by one skilled in the art. For example, anti-DSC2 antibodies can
be administered to living bodies in an amount such that cytotoxicity
based on effector function against DSC2-expressing cells can be
confirmed. For example, although there is a certain amount of difference
depending on symptoms, anti-DSC2 antibody dosage is 0.1 mg to 250 mg/kg
per day. Usually, the dosage for an adult (of weight 60 kg) is 5 mg to
17.5 g/day, preferably 5 mg to 10 g/day, and more preferably 100 mg to 3
g/day. The dosage schedule is from one to ten times over a two to ten day
interval, and for example, progress is observed after a three to six
times administration.

VI. Immunogenic Compositions

[0176]According to the present invention, it was discovered that the
administration of anti-DSC2 antibody damages cancer cells through the
effector function of the antibody. Therefore, the present inventors
considered that a composition inducing DSC2 antibodies with effector
function has equivalent therapeutic effects with the present
pharmaceutical composition comprising an antibody with effector function.
It is expected that such vaccinating effect can be achieved by
administering DSC2 polypeptide, or a nucleic acid molecule that expresses
the polypeptide. Thus, the present invention provides immunogenic
compositions for inducing antibodies with at least one effector function
against DSC2-expressing cells in vivo. The compositions typically
comprise as an active ingredient, a DSC2 polypeptide, or a nucleic acid
molecule that expresses the polypeptide. It is preferred that the
polypeptide or the nucleic acid molecule is an isolated or purified
substance.

[0177]The immunogenic compositions of the present invention are
particularly useful in vaccine therapy against diseases associated with
DSC2-expressing cells.

[0178]The immunogenic compositions of the present invention are effective
as, for example, vaccine compositions for lung, colon, pancreatic,
prostate, breast, gastric or liver cancer therapies. They may be used
against humans and other animals, including mice, rats, guinea pigs,
rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons, and
chimpanzees.

[0179]The DSC2 polypeptide included in the present immunological
composition may be either the whole DSC2 protein or a fragment thereof so
long as the fragment retains the ability to induce in vivo antibodies
that recognize DSC2 and have effector function. Herein after, such
fragments will be called immunologically active fragments.

[0180]The DSC2 polypeptide can be derived from any species, preferably
from a mammal such as a human, mouse, or rat, and more preferably from a
human to be treated with the composition through conventional
purification techniques. Moreover, the nucleotide and amino acid
sequences of human DSC2 are known (cDNA nucleotide sequence of DSC2 type
2b (GenBank Accession No. NM--004949; SEQ ID NO: 1) and DSC2 type 2a
(GenBank Accession No. NM--024422; SEQ ID NO:2), and the
corresponding amino acid sequences are described in SEQ ID NOs: 3
(GenBank Accession No. NP--004940) and 4 (GenBank Accession No.
NP--077740), respectively). Thus, to obtain the DSC2 polypeptide, a
person may chemically synthesize or genetically produce the polypeptide
based on these sequence information. For example, one skilled in the art
can routinely isolate or construct a polynucleotide comprising the
objective nucleotide sequence, insert the gene into a suitable expression
vector to transform a suitable host cell, and obtain a protein comprising
the target amino acid sequence by culturing the host cell under suitable
conditions for expression of the polypeptide from the cells or the
culture supernatant.

[0181]An immunologically active fragment of the whole DSC2 protein may
also be prepared based on the above sequence information. Since the
region of positions 1 to 32 of the N-terminus of DSC2 is predicted to
correspond to a signal sequence (Greenwood M D. et al., Genomics 44(3):
330-5, 1997 Sep. 15), it is preferred to avoid this region for the
fragment. The extra-cellular domains (position 144 to 540 of DSC2) are
particularly preferred as the immunologically active fragment to be
included in the present composition. However, the present invention is
not restricted thereto, and much shorter polypeptides may be adopted as
the immunologically active fragment.

[0182]Furthermore, the DSC2 polypeptide may be a protein which has been
modified from the natural occurring DSC2 protein so long as the modified
molecule has the ability to induce in vivo antibodies that recognize DSC2
and have effector function. Such modifications include those mentioned
above for the antibody of the present invention.

[0183]In addition to the immunogenic proteins (whole DSC2 polypeptides,
immunologically active fragments thereof, and modified molecules
thereof), the present immunogenic composition may comprise
pharmaceutically acceptable carriers. Similar substances to those
mentioned for the pharmaceutical composition of the invention may be
employed as pharmaceutically acceptable carriers for the present
immunogenic composition. If necessary, the compositions can also be
combined with an adjuvant. Killed tuberculosis bacteria, diphtheria
toxoid, saponin and the like can be used as the adjuvant.

[0184]Alternatively, DNAs coding for the immunogenic proteins, or cells
retaining those DNAs in an expressible state, can be used as the active
ingredient of the present immunogenic compositions. Methods for using
DNAs expressing the target antigen as immunogens, so-called DNA vaccines,
are well known in the art. For example, DNA vaccines can be obtained by
inserting a DNA encoding a whole DSC2 polypeptide, immunologically active
fragment thereof, or a modified molecule thereof into an appropriate
expression vector.

[0185]Retrovirus vectors, adenovirus vectors, adeno-associated virus
vectors, Sendai virus vectors or such can be used as the vector. In
addition, DNAs in which a DNA encoding an immunogenic protein is
functionally connected downstream of a promoter can be directly
introduced into cells as naked DNA, and then expressed. Naked DNA can be
encapsulated in ribosomes or viral envelope vectors and introduced into
cells.

[0186]When DNAs encoding the immunogenic proteins, or cells transformed
with the same are used as immunogenic compositions of the present
invention, they can be combined with immunogenic proteins as well as
carrier proteins that enhance their immunogenic properties. For more
details, the explanation on pharmaceutical compositions containing
nucleic acids comprising sequences encoding antibodies or functional
derivatives thereof can be referred.

[0187]Whether a given polypeptide or polynucleotide induces antibodies
against the polypeptide in vivo can be determined by actually immunizing
an animal, and confirming the activity of the induced antibodies.
Similarly, the induction of anti-tumor immunity by a polypeptide can be
confirmed by observing the induction of antibody production against
tumors in an animal immunized with the polypeptide. For example, when
antibodies against a polypeptide are induced in a laboratory animal
immunized with the polypeptide, the suppression of tumor cell growth
indicates the ability of the polypeptide to induce anti-tumor immunity.
The ability for antibody induction and confirmation of the property of
the induced antibody of a polypeptide can be carried out, for example,
using methods described in Examples.

VII. Induction of Immune Response

[0188]Moreover, the present invention provides a method for inducing
antibodies with at least one effector function against DSC2-expressing
cells in vivo. Specifically, the method comprises administering the
aforementioned immunogenic composition of the present invention to a
subject. For example, each day, 0.1 mg to 250 mg per kilogram of the
immunogenic composition of the present invention can be administered
orally or parenterally. Parenteral administration includes subcutaneous
injection and intravenous injection. The administrative dose for a single
adult is usually 5 mg to 17.5 g/day, preferably 5 mg to 10 g/day, and
more preferably 100 mg to 3 g/day.

[0190]Furthermore, the DSC2 polypeptide and nucleic acid molecules
encoding the polypeptide can also be used for the induction of immune
response in vivo other than the production of antibodies against the DSC2
polypeptide. Namely, it is known that cytotoxic T lymphocytes (CTL)
specific for a protein can be induced by presenting the protein to a T
cell via an antigen presenting cell (APC) either in vivo or ex vivo.
Thus, similarly, the DSC2 polypeptide may be presented to T cells either
in vivo or ex vivo for the induction of CTL. It is preferred to use
purified or isolated polypeptides or nucleic acid molecule for such
induction of immune response.

[0191]For example, patient blood cells e.g., peripheral blood mononuclear
cells (PBMC) are collected, transformed with a vector that expresses an
immunogenic protein, and returned to the patient. Transformed blood cells
produce the immunogenic protein inside the body of the patient, and
induce objective antibodies.

[0192]Alternatively, PBMCs of the patient are collected, the cells are
contacted with the immunogenic protein ex vivo, and following the
induction of APCs or CTLs, the APCs or CTLs may be administered to the
subject. Further, if needed, APCs or CTLs induced in vitro can be cloned
prior to administration. By cloning and growing cells having high
activity of damaging target cells, cellular immunotherapy can be
performed more effectively. Furthermore, APCs and CTLs isolated in this
manner may be used for cellular immunotherapy not only against
individuals from whom the cells have been derived, but also for other
individuals with similar types of tumors.

[0193]Generally, when using a polypeptide for cellular immunotherapy,
efficiency of the CTL-induction is known to be increased by combining a
plurality of polypeptides having different structures and contacting them
with APCs, particularly, dendritic cells. Therefore, when stimulating
APCs with protein fragments, it is advantageous to use a mixture of
multiple types of fragments.

[0194]All prior art references cited herein are incorporated by reference
in their entirety.

[0201]Below, the present invention is further explained based on Examples.
However, materials, methods and such described therein only illustrate
aspects of the invention and in no way are intended to limit the scope of
the present invention. As such, materials, methods and such similar or
equivalent to those described therein may be used in the practice or
testing of the present invention.

(1) Cell Lines

[0202]Human lung, colon, pancreatic, prostate, breast, gastric and liver
cancer cell lines were propagated as a monolayer in an appropriate medium
supplemented with 10% fetal bovine serum. The cell lines used in the
experiment are shown in Table 1.

[0204]According to standard protocols, individual protein specific
polyclonal antibodies were produced using His-tagged fusion proteins
expressed in bacteria as immunogens. These fusion proteins comprised a
protein portion that corresponded to a specific portion of the protein
(residues 144 to 540).

[0207]To produce the recombinant protein of DSC2, 293T cells were
transiently transfected with the plasmid pQC/DSC2 mH/IPG, using
Lipofectamine 2000 (Invitrogen) according to the manufacturer's
instructions, and after 48 h incubation, the 293T cells were harvested.

[0208]On the other hand, to produce the secretory form recombinant protein
DSC2-s, a cell line was established using Pantropic Retroviral Expression
System (Clontech) according to the manufacturer's instructions.
Specifically, GP2-293 cells were co-transfected with pQC/DSC2-s/IPG and
pVSV-G (Clontech). After 48 h incubation, virus-containing supernatants
were centrifuged. The retrovirus vector solution was prepared by
resuspending the precipitation with TNE solution (Tris-HCl, pH7.8, 130 mM
NaCl, 1 mM EDTA). 293T cells were transfected with pQC/DSC2-s/IPG using
the retrovirus vector solution diluted by 8 μg/mL hexadimethrine
bromide (SIGMA)-containing DMEM supplemented with 10% FBS. The selection
of pQC/DSC2-s/IPG-transfected 293T cells, DSC2-s/293T, was performed
using 5 μg/mL puromycin (SIGMA). The His-tagged proteins in
DSC2-s/293T-culture supernatants were purified using TALON Purification
kit (Clontech).

[0210]After three immunizations with 2-day interval, respectively, the
mice were immunized with DSC2-transfected 293T cells or DSC2-s purified
antigen in PBS. Then, cells from the lymph node of immunized mice were
harvested and fused with myeloma cell line, P3U1. The hybridomas were
subcloned by selection using flow cytometry and subsequent single-cell
cloning by limiting dilution. Antibody in cell culture supernatants of
isolated hybridomas was confirmed by Immunoprecipitation analysis.
Antibody-containing supernatants from positive clones were tested by
ELISA for the relative binding affinity against the DSC2 extracellular
domain expressed on DSC2 over-expressing cell line, H358. The antibody
against DSC2 (representing amino acids 1 to 901) was designed 48-5, and
that against DSC2-s (representing amino acids 1 to 688) as s10-4.

(2-3) Humanized Chimeric Antibodies

[0211]Humanized chimeric antibodies ch48-5 and chs10-4 based on the mouse
monoclonal antibodies 48-5 and s10-4, respectively, were prepared
according to previously reported methods (Alvin Y Liu et al., Proc Natl
Acad Sci USA 84: 3439-43, 1987; Mitchel E Reff et al., Blood 83(2):
435-45, 1994). Specifically, total RNA was extracted from mouse 48-5 or
s10-4 hybridoma cells by RNeasy Mini Kit (QIAGEN, 74104), and then
reverse-transcribed to single-stranded cDNA using GenenRacer® kit
(Invitrogen, L1502-02). Gene encoding the variable region of the antibody
(Fab) was determined by PCR using this cDNA as template and the following
set of primers:

[0213]Genes corresponding to each of the variable regions were amplified
by PCR and cloned into an antibody expression cassette vector using a
NotI-BamHI brachet. A retrovirus vector wherein the expression of the
variable region gene is controlled by the CMV promoter was used as the
antibody expression cassette vector. The vector for expressing the
H-chain contained the hygromycin resistance gene (SEQ ID NO: 85), and
that for the L-chain contained the puromycin resistance gene (SEQ ID NO:
86).

[0214]The vectors expressing the H-chain and L-chains were co-transfected
into Chinese hamster ovary (CHO) cells for 48-5 and s10-4, respectively.
The cells were selected using F-12 medium containing 500 μg/ml
hygromycin and 10 μg/ml puromycin, the medium was exchanged with
serum-free medium (CHO--S--SFM; GIBCO, 12052-098), and the chimeric
antibody contained in the culture supernatant was purified via Protein A
column.

(2-4) Human Antibodies

(i) Screening of Phase Expression Libraries Using Culture Cells

[0215]The screening of human scFV antibody against DSC2 was achieved using
a phage library encoding human scFV antibodies created in the Institute
for Antibodies (IFA; Nagoya, Japan). Specifically, the screening of human
scFV antibody against DSC2 was performed using phage library AIMS4 coding
for human scFV antibodies (WO 01/062907) following the method described
in JP-A 2005-185281.

[0216]More specifically, the 1st screening was conducted as follows:
MIAPaca-2 cells showing high expression of DSC2 were cultured on 15 cm
dishes, harvested with the addition of 2 mg/ml collagenase I and a cell
dissociation buffer (both Gibco BRL), and washed with cooled PBS. A
solution of human antibody phage library (2×10113 cfu) was
mixed with 4×107 of the cells, BSA and NaN3/MEM were
added at final concentrations of 1% and 0.1%, respectively, and the final
volume was adjusted to 1.6 ml. The mixture was gently agitated for 4 hrs
at 4° C., dispensed at equal volumes into two tubes, poured onto
organic solution (dibutyl phthalate:cyclohexane=9:1) and centrifuged at
3,000 rpm for 2 min. The supernatant was removed, the pellet (cells) were
resuspended in 0.7 ml of 1% BSA/MEM, and centrifuged on equal volume of
low polarity solvent. This step was repeated twice. The supernatant was
removed, the cells were resuspended in 0.3 ml of PBS, frozen with liquid
nitrogen, and melted at 37° C. to obtain phages within the cells.

[0217]These phages were allowed to infect 20 ml of E. coli DH12S (OD=0.5)
for 1 hr. The infected cells were transferred into 600 ml of 2×YTGA
medium (2×YT, 200 μg/ml ampicillin sulfate, 1% glucose), and
cultured overnight at 30° C. A 10 ml aliquot thereof was added to
200 ml of 2×YTA medium (2×YT, 200 μg/ml ampicillin
sulfate) and cultured for 1.5 hrs at 37° C. After additional
incubation, 1×1011 of helper phage KO7 was added and further
cultured for 1 hr at 37° C. 800 ml of 2×YTGAK (2×YT,
200 μg/ml ampicillin sulfate, 0.05% glucose, 50 μg/ml kanamycin)
were added and cultured overnight at 30° C. The culture was
centrifuged at 8,000 rpm for 10 min, the supernatant was mixed with 200
ml of PEG liquid (20% polyethylene glycol 6000, 2.5M NaCl) and
centrifuged at 8,000 rpm for 10 min. The phages are contained in the
pellet, and the pellet was suspended in 10 ml of PBS and a portion
thereof was used for examining the number of E. coli infected with the
phage.

[0218]The second screening was performed similarly to the first screening
using 0.8 ml of reactive solution (1% BSA, 0.1% NaN3/MEM),
2×107 culture cells, and 1×1010 phages screened in
the first screening, wherein the total volume of the mixture was half of
that used in the first screening.

[0219]The third screening was performed similarly to the second screening
except that 2×107 of 293T cells transfected with DSC2 and the
phages screened in the second screening were used.

(ii) DNA Sequencing and Expression Confirmation

[0220]The screened E. coli was diluted and cultivated on nutrient agar
supplemented with 100 μg/ml of ampicillin. Obtained colonies were
picked up and incubated overnight at 30° C. in 2×YTGA
medium. [0221]1. For sequencing, DNA was obtained from the culture with
PI-50 (Kanebo), and the nucleotide sequence was determined by the dideoxy
method. [0222]2. The expression of the protein was detected as the
expression of cp3 fusion protein. Specifically, 0.05 ml of the culture
was added to 1.2 ml of 2×YTAI (2×YT, 200 μg/ml ampicillin
sulfate, 0.5 mM IPTG) and incubated at 30° C. The supernatant was
collected by centrifugation at 15,000 rpm for 5 min, and reacted on
Maxisorp® high protein-binding capacity ELISAplate (NUNC) for 2 hrs at
37° C. After aspirating the solution on the plate, the antibody on
the plate was blocked with 5% BSA for 2 hrs at 37° C., and the
blocking solution was removed. Rabbit anti-cp3 antibody (MBL) diluted to
1:2,000 with 0.05% Tween/PBS was added to the plate and reacted at room
temperature for 1 hr, and the plate was washed with PBS. Similarly, HRP
tagged goat anti-rabbit IgG antibody (MBL) diluted to 1:2,000 with 0.05%
Tween/PBS was added to the plate, reacted at room temperature for 1 hr.
and the plate was washed with PBS. 100 μl of OPD solution was added to
the plate and reacted at room temperature for 15 min, the reaction was
quenched by the addition of 2M ammonium sulfate. The fusion protein was
detected by measuring the absorbance at 492 nm with SPECTRAmax340PC
(Molecular Devices).(iii) Flow Cytometry

[0223]Flow cytometry analysis confirmed that two clones of human scFV
antibody, clones 332 and 545, positively reacted to the antigen. These
two clones consisted of the following amino acid sequences:

These human scFV antibodies were converted to complete IgG forms at
IFA.(3) Semiquantitative RT-PCR for DSC2 and c-erbB2

[0224]Total RNA was extracted from the cell lines using the RNeasy®
Kit (QIAGEN). In addition, mRNA was purified from total RNA by Oligo
(dT)-cellulose column (Amersham Biosciences) and converted into
first-strand cDNA by reverse transcription (RT) using SuperScript
First-Strand Synthesis System (Invitrogen). Appropriate dilutions of each
first-stranded cDNA were prepared for subsequent PCR amplification by
monitoring GAPDH as a quantitative control. The primer sequences used
were as follows:

[0225]All PCR reactions involved initial denaturation at 94° C. for
2 min, cycles of 94° C. for 30 s, 58° C. for 30 s, and
72° C. for 1 min, and annealing step, which were conducted on
GeneAmp PCR system 9700 (PE Applied Biosystems). The reactions included
21 and 32 cycles for GAPDH and c-erbB2, respectively, and the annealing
temperature was lowered gradually from 62° C. to 58° C. for
these genes. For β-actin, the reaction included 20 cycles and the
annealing temperature was lowered gradually from 62° C. to
57° C., which was 30 cycles and lowering gradually from 62°
C. to 56° C. for DSC2.

[0226]The over-expression of DSC2 was found in lung cancer cell line
NCI-H358 (FIG. 1A). In addition, to elucidate the efficacy of anti-DSC2
polyclonal antibody (BB049) on various cancers, the expression of DSC2
was confirmed. The over-expression of DSC2 was detected in colon cancer
cell line HT-29, pancreatic cancer cell line KLM-1, prostate cancer cell
line LNCap FGC, breast cancer cell line T47D, gastric cancer cell line
MKN7, and liver cancer cell line HepG2 (FIG. 1B-G).

(4) Flow Cytometric Analysis

[0227]Cancer cells (5×106) were incubated at 4° C. for
30 min with the purified polyclonal antibody (pAb: BB049), monoclonal
antibodies (mAb), rabbit IgG (the control for pAb) or mouse IgG (the
control for mAb). The cells were washed with phosphate buffer solution
(PBS) and then incubated at 4° C. for 30 min in FITC-labeled Alexa
Fluor 488. The cells were washed again in PBS, and analyzed on flow
cytometer (FACSCalibur®, Becton Dickinson) and then analyzed by BD
CellQuest® Pro software (Becton Dickinson). Mean fluorescence
intensity (MFI) was defined as a ratio of the flow cytometric intensity
(intensity by each protein specific antibody/intensity by rabbit IgG).

[0228]Using DSC2 over-expressing cells, the binding proportions of
anti-DSC2 antibodies on the cell surface were investigated. As a result,
binding proportions of anti-DSC2 polyclonal antibody BB049 on NCI-H358,
HT-29, KLM-1, LNCap FGC, T47D, MKN7, and HepG2 cell surfaces (MFI: 82.8,
56.8, 47.8, 15.8, 92.2, 51.8, and 20.7, respectively) were higher than
that of rabbit IgG (control). Further, binding proportions of anti-DSC2
mouse monoclonal antibodies 48-5 and s10-4 on NCI-H358 cell surface (MFI:
10.0 and 11.1, respectively) were higher than that of mouse IgG
(control), those of anti-DSC2 human-mouse chimeric antibodies ch48-5 and
chs10-4 on NCI-H358 cell surface (MFI: 34.3 and 54.8, respectively)
were higher than that of mouse IgG (control), and those of anti-DSC2
human antibodies 332 and 545 on NCI-H358 cell surface (MFI: 9.48 and
5.52, respectively) were higher than that of human IgG (control).

(5) ADCC Assays

[0229]Target cells were exposed to 0.8 μM of calcein acetoxymethyl
estel (Calcein-AM, DOJINDO) at 37° C. for 30 min. Calcein-AM
becomes fluorescent after the cleavage of calcein-AM by cellular
esterases that produce a fluorescent derivate calcein. Target cancer
cells were washed twice before being added to the assay, and then plated
on 96-well U-bottom plates (4×103 cells/well). Human
peripheral blood mononuclear cells (PMBC) were harvested from healthy
person, separated using Ficoll-Paque (Amersham Biosciences) density
gradient centrifugation, and then used as effector cells. Target cancer
cells (T) and effector cells (E) were co-incubated in 250 μl of AIM-V
medium in 96-well plates at various E:T ratios (200:1, 100:1, 50:1, 25:1,
12.5:1, and 6.25:1) with BB049 anti-DSC2 polyclonal antibody (2
μg/well) or control antibody Herceptin (2 μg/well, Roche). This
incubation was carried out in triplicate, in 250 μL of AIM-V medium
(Life Technologies, Inc), at 37° C. for six hrs. Control assays
included the incubation of target cells with anti-DSC2 polyclonal
antibody BB049 or effector cells alone. Herceptin was used as a control
in some of the experiments.

[0230]The ADCC effects of anti-DSC2 polyclonal antibody (BB049) for these
cells were evaluated based on the fluorescent images of viable cells that
could be rapidly acquired using the IN Cell Analyzer 1000 (Amersham
Bioscience). These images were numerically converted as viable cell
counts (cell area for MKN7) by counting the fluorescent object or using
Developer tool ver.5.21 software (Amersham Bioscience).

[0231]Herceptin was used as a control in several experiments (FIGS. 2A and
2B). Direct cell damage of NCI-H358, HT-29, KLM-1, LNCap FGC, T47D, MKN7,
and HepG2 cells by BB049 anti-DSC2 polyclonal antibody itself was not
observed. However, BB049 induced ADCC in NCI-H358, HT-29, KLM-1, LNCap
FGC, T47D, MKN7, and HepG2 cells that over-expressed DSC2 (FIG. 3A-G),
while it caused no effect on SK-LU-1 cells with DSC2 low-expression (FIG.
3H).

[0232]The ADCC effects of anti-DSC2 mouse monoclonal antibodies 48-5 and
s10-4 on DSC2 over-expressing cell line NCI-H358 were estimated. As
described above, target and effector cells were prepared. Under the
condition at an E:T ratio of 100:1, at various concentrations (0, 0.5,
1.0, 5.0, 10.0, and 21.0 μg/well for 48-5; and 0, 0.5, 2.0, 8.0, 16.0,
and 35.0 μg/well for s10-4), 48-5 and s10-4 induced ADCC in NCI-H358
cells (FIG. 4). No direct cell damage of NCI-H358 cells by anti-DSC2
monoclonal antibodies (48-5 and s10-4) was observed.

[0233]The ADCC effects of anti-DSC2 human-mouse chimeric antibodies ch48-5
and chs10-4 on DSC2 over-expressing cell line NCI-H358 were estimated. As
above, target and effector cells were prepared. Under the condition at an
E:T ratio of 100:1, at various concentrations (0, 0.05, 0.1, 5.0, and
0.15 μg/well), ch48-5 and chs10-4 induced ADCC in NCI-H358 cells (FIG.
5). No direct cell damage of NCI-H358 cells by anti-DSC2 human-mouse
chimeric antibodies (ch48-5 and chs10-4) was observed.

[0234]The ADCC effects of anti-DSC2 human antibodies 332 and 545 on DSC2
over-expressing cell line NCI-H358 were estimated. As above, target and
effector cells were prepared. Under the condition at an E:T ratio of
100:1, at various concentrations (0.001, 0.01, 0.1, 1.0, 10, and 100
μg/well), 332 and 545 induced ADCC in NCI-H358 cells (FIG. 6). No
direct cell damage of NCI-H358 cells by anti-DSC2 human antibodies (332
and 545) was observed.

INDUSTRIAL APPLICABILITY

[0235]The present invention is based, at least in part, on the discovery
that DSC2-expressing cells can be damaged by utilizing the cytotoxicity
of antibodies. Strong expression of DSC2 gene was identified by the
present inventors in lung, colon, pancreatic, prostate, breast, gastric
and liver cancers. Herein, results demonstrating the effect of antibody
dependent cell-mediated cytoxicity (ADCC) of anti-DSC2 antibodies on
lung, colon, pancreatic, prostate, breast, gastric and liver cancer cell
lines are presented. Thus, the antibodies, compositions and methods of
the present invention provide a novel approach for treating diseases
associated with DSC2-expression, for example, lung, colon, pancreatic,
prostate, breast, gastric and liver cancers.

[0236]While the invention has been described in detail with reference to
specific embodiments thereof, it will be apparent to those skilled in the
art that various changes and modifications can be made therein without
departing from the spirit and scope of the invention, the metes and
bounds of which are set by the appended claims.